This invention relates to a device for electrophoresis used for analyzing an extremely small quantity of protein or nucleic acid at a high speed and with a high resolution.
It has been known to analyze an extremely small quantity of protein or nucleic acid by electrophoresis, and a slab gel device has been a representative example of a device using this method of analysis. A slab gel device comprises a pair of glass slabs with the space therebetween filled with a gel to form a migration area. After a sample is injected at one end of the gel portion, a voltage difference of about 100V is applied at both its ends to cause the electrophoresis of the target substance for development. The developed target substance may then be detected by exposing the gel to a beam of laser light and measuring its absorption, by using a dye or by preliminarily labeling the target substance with a radioactive isotope and then using an auto-radiograph.
When a slab gel device is used, however, only a moderate voltage difference can be applied for analysis because the generation of Joule heat inside the gel presents a problem. For this reason, a long migration time from several hours to nearly 20 hours is required, and hence this cannot be considered a useful device when a quick analysis is required such as in a DNA analysis.
In view of the above, it is being proposed to make use of a capillary device for electrophoresis with a buffer filling the interior of a glass capillary having inner diameter of about 100 xcexcm or less and a high voltage difference applied across its both ends after a sample has been injected at one end so as to cause the target substance to be developed inside the capillary. FIG. 1 shows a prior art example of such a capillary device for electrophoresis, having a glass capillary 20 with both of its ends immersed in a migration buffer 22a or 22b contained in a buffer tank 21a or 21b. Electrodes 23a and 23b, respectively connected to the plus and minus terminals of a high-voltage source 24 are immersed individually in these buffer tanks 21a and 21b. A detector 25 for detecting the target substance migrating inside the glass capillary 20 is disposed on one side of the capillary 20. The target substance is developed inside the glass capillary 20 and detected by applying a high voltage difference across the glass capillary 20 from the high voltage source 24 through the electrodes 23a and 23b and the buffer 22a and 22b. A device thus structured is useful because its surface area is large compared to the volume inside the glass capillary 20 and hence it can be cooled efficiently. Since the heat generated by the application of a high voltage can thus be radiated away quickly, a very small quantity of a DNA sample, for example, can be quickly analyzed with a high resolution by applying a high voltage.
Glass capillaries used in such a device for electrophoresis are not easy to handle when, for example, they are to be exchanged for replacement by the user since their outer diameter is as small as 10-100 xcexcm and hence they are easily damaged. In view of the above, D. J. Harrison et al. proposed a planar capillary member formed by joining two planar substrates (Anal. Chim. Acta., 238 (1993) 361-366). FIG. 2 shows an example of such a planar capillary member having a first substrate 1 a with a primary groove for analysis 8, a secondary groove for sample injection 7 and buffer tanks 9 formed thereon by a photo-fabrication technology and a second substrate 1b with throughholes 10 formed therethrough by a ultrasonic technology at positions corresponding to the buffer tanks 9. These two substrates 1a and 1b are joined together to form a planar capillary member.
When an analysis is carried out with such a planar capillary member, a buffer is introduced into the grooves 7 and 8 and a sample is injected from the buffer tank 9 at one end of the secondary groove 7. A high voltage is applied to the electrodes preliminarily provided on the inner walls and their neighborhoods of the throughholes 10 corresponding to the buffer tanks 9 at both ends of the secondary groove 7, thereby leading the sample by electrophoresis to the crossing point between the grooves 7 and 8. Thereafter, a high voltage is applied to the electrodes preliminarily provided on the inner walls and their neighborhoods of the throughholes 10 corresponding to the buffer tanks 9 at both ends of the primary groove 8 such that the target substance is developed therein. The developed target substance may be detected, for example, by applying a beam of laser light from outside and measuring its absorptivity.
Planar capillary members thus structured are not easily damaged because of their planar shape and are easier to handle than ordinary glass capillaries. With the detection method by light absorption by such a planar capillary device of electrophoresis, however, the sensitivity of detection becomes lower if the quantity of the sample is extremely small.
It is therefore an object of this invention to provide a planar capillary device with which an extremely small quantity of substance in the capillary can be measured with a high sensitivity.
A component of a device for electrophoresis embodying this invention, with which the above and other objects can be accomplished, may be characterized as comprising a pair of planar members such as glass substrates joined together one on top of the other such that a capillary is formed by a groove on the surface of at least one of these planar members, a metallic film on the inner wall of at least a portion of the capillary thus formed between the pair of planar members and a transparent prism disposed over this metallic film. A device embodying this invention for electrophoresis incorporating such a component may be characterized as additionally including electrodes inserted at selected positions in the capillary for providing a voltage difference between them, a light detector and a data processor. The light source is disposed to cause light to be made incident onto the metallic film at angles of incidence greater than the critical angle for total reflection, and the detector serves to detect reflected light. The data processor receives detection signals from the detector and obtains therefrom a resonance curve for optical excitation of surface plasmons on the metallic film. It also stores data on separation times of known components in electrophoresis for reference and serves to detect changes in the resonance curve to thereby analyze the components of a sample. The positions at which the electrodes are inserted are selected so as to prevent the metallic film provided inside the capillary from becoming dissolved or otherwise affected adversely by the voltages applied to the electrodes.